WO2021237270A1 - Procédé de traitement de la leucémie - Google Patents

Procédé de traitement de la leucémie Download PDF

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WO2021237270A1
WO2021237270A1 PCT/AU2021/000038 AU2021000038W WO2021237270A1 WO 2021237270 A1 WO2021237270 A1 WO 2021237270A1 AU 2021000038 W AU2021000038 W AU 2021000038W WO 2021237270 A1 WO2021237270 A1 WO 2021237270A1
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mir
agent
inhibitor
mdm2
expression
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PCT/AU2021/000038
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Timothy John MOLLOY
David Dang Fung Ma MA
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St Vincent's Hospital Sydney Limited
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Definitions

  • the present disclosure relates to a method for treating a haematological malignancy or pre- malignant condition in a subject.
  • the method involves administering to a subject with Acute Myeloid Leukaemia (AML), an effective amount of a first agent comprising an MDM2/MDMX inhibitor, a second agent comprising an inhibitor of miR-10a expression and/or activity, and optionally, a chemotherapeutic agent such as a "standard of care" (SOC) chemotherapeutic agent for AML (eg cytarabine).
  • AML Acute Myeloid Leukaemia
  • a chemotherapeutic agent such as a "standard of care" (SOC) chemotherapeutic agent for AML (eg cytarabine).
  • SOC standard of care
  • Acute myeloid leukaemia otherwise known as acute myelogenous leukaemia and acute granulocytic leukaemia amongst others, is a rare disease that affects the blood and bone marrow.
  • AML acute myeloid leukaemia
  • the disease is typically curable in 35% of cases, but in patients over the age of 60 years, the disease is only curable in about 10% of cases [2], And for those older patients who are unable to tolerate intensive chemotherapy (eg using the standard of care chemotherapeutic agent, cytarabine), the typical survival period is only 5-10 months [2],
  • p53 is a critical tumour suppressor protein that has a central role in modulating diverse cellular processes including proliferation, cell cycle, DNA repair, senescence, apoptosis and autophagy [1], Approximately half of all human cancers bear inactivating mutations of p53, and p53-deficent mice develop a range of tumours with high penetrance [2], While common in solid cancers, TP53 mutations are less frequent in haematological malignancies such as AML, in which they are present in only around 5% of patients [3], In the majority of AML cases however, it is thought that p53 is repressed by other means, including the overexpression of its two primary negative regulators, murine double minute 2 homolog (MDM2) and murine double minute 4 homolog (MDM4; otherwise known as MDMX) [4] [5], or via mutation of the nucleophosmin 1 (NPM1) [6] which occurs in around 30% of AML patients [7], As a nucleocytoplasmic shuttle
  • miR-10a is strongly overexpressed in AML compared to healthy bone marrow (up to 80-fold), and also displays subgroup-specific expression [13], miR-10a is involved in the p53/Rb tumour suppressor/stress response pathway regulation via its repression of a number of the p53/Rb networks' key genes, including MDM4, Rb regulator RB-Inducible Coiled-Coil 1 (RBCC1), and p21 regulator Transcription Factor AP2-Gamma (TFAP2C) [13] [14].
  • MDM4 Rb regulator RB-Inducible Coiled-Coil 1
  • TFAP2C p21 regulator Transcription Factor AP2-Gamma
  • miR-10a may have a role in the response to MDM2 inhibitors in AML and could represent both a valuable therapeutic target for modulating drug responses via de-repression of its key p53/Rb-related targets, and a biomarker of response.
  • this disclosure relates to a method of treating a haematological malignancy or pre-malignant condition in a subject, said method comprising administering to said subject an effective amount of:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity; and (ii) optionally, a chemotherapeutic agent.
  • the method is preferably applied to the treatment of AML, more preferably, to the treatment of AML overexpressing miR-10a.
  • the first agent comprising an inhibitor of MDM2 and/or MDMX is preferably selected from cis- imidazoline compounds such as Nutlin-1, Nutlin-2 and Nutlin-3 (ie ( ⁇ )-4-[4,5-Bis(4-chlorophenyl)-2-(2- isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-l-carbonyl]-piperazin-2-one) [62] [63], and especially the (-)-Nutlin 3 enantiomer known as Nutlin-3a, or a derivative or analogue thereof.
  • cis- imidazoline compounds such as Nutlin-1, Nutlin-2 and Nutlin-3 (ie ( ⁇ )-4-[4,5-Bis(4-chlorophenyl)-2-(2- isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-l-carbonyl]-piperazin
  • the second agent comprising an inhibitor of miR-10a is preferably selected from anti- microRNA-lOa agents comprising a single-stranded antisense oligonucleotide designed to specifically target mature miRNA 10a.
  • the chemotherapeutic agent may be selected from those chemotherapeutic agents that may be regarded as "standard” in the art or comprise what is considered a “standard of care” chemotherapeutic agent (eg cytarabine, or cytarabine in combination with daunorubicin).
  • composition comprising:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • the present disclosure relates to a kit comprising first, second and third containers (eg vials), wherein the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX, the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity, and the third container contains a chemotherapeutic agent; optionally packaged with instructions for the use of the kit in the method of the first aspect
  • the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX, and the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity; optionally packaged with instructions for the use of the kit in the method of the first aspect.
  • first and second containers eg vials
  • the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX
  • the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the present disclosure relates to a method of predicting effectiveness of MDM2 and/or MDMX inhibition in treating an individual with a malignant condition (preferably a haematological malignancy such as AML) or pre-malignant condition, comprising quantifying miR-10a expression and/or modulation of downstream genes/factors (eg in a suitable body sample (eg a blood or bone marrow sample).
  • a malignant condition preferably a haematological malignancy such as AML
  • pre-malignant condition comprising quantifying miR-10a expression and/or modulation of downstream genes/factors (eg in a suitable body sample (eg a blood or bone marrow sample).
  • Figure 1 provides the results of an experiment designed to assess whether miR-10a expression is correlated to cancer cell line drug sensitivity, specifically for the MDM2 inhibitor, Nutlin-3a
  • the endogenous expression of miR-10a in 60 cancer cell lines was correlated to sensitivity to 265 drugs (A) and Nutlin-3a was found to be the 4th strongest correlation (B);
  • B Cell lines with endogenous expression below the median level had a mean IC50 of 34.1 ⁇ , versus 95.7 ⁇ for those above;
  • Figure 2 provides the results of an experiment designed to assess whether expression of miR-10a downstream target genes TFAP2C, CDKNIA, TPS 3 and MDM4 are correlated to sensitivity to the MDM2 inhibitor, milademetan (DS-3032b) in 38 pre-treatment AML primary patient samples treated ex vivo.
  • the IC50 dose was calculated for each sample, enabling them to be split into "resistant” (samples with the top 33% of IC50 values) and "sensitive” (bottom 33%) subgroups.
  • Resistant samples had a 21.9- fold higher mean IC50 than sensitive samples (p ⁇ 0.001): (A) Samples sensitive to DS-3032b expressed significantly higher levels of TFAP2C, TP53 and MDM4 mRNA, and significantly lower levels of CDKNIA; (B) These patients also participated in a Phase I/II clinical trial of DS-3032b and response data was available for the cohort.
  • Responders ie those that experienced either complete remission (CR), complete remission with incomplete platelet recovery (CRp) or haematological improvement (HI) displayed analogous expression patterns of miR-10a targets compared to non-responders;
  • F-H miR-10a itself was also strongly predictive of outcome in patients treated with cytarabine-based chemotherapy + HSCT.
  • Figure 3 provides the results of experiments designed to assess whether miR-10a mediates sensitivity to Nutlin-3a and cytarabine in AML.
  • A-B miR-10a inhibition in the miR-10a-high AML cell lines OC1-AML3 and IMS-M2 resulted in significant sensitisation to Nutlin-3a (A) and cytarabine (B).
  • C-D Conversely, overexpression of miR-10a in miR-10a-low AML cell lines MV4-11 and HL60 led to significant resistance.
  • E-F The exogenous expression of Rblccl (OE) or knockdown (KD) of p21 alone was sufficient to recapitulate these effects in OCI-AML3 and 1MS-M2 cells, confirming that these miR- 10a targets are mediators of Nutlin-3a and cytarabine sensitivity.
  • G-H OCI-AML3 cells transduced with a miR-10a or scrambled control inhibitor (G) and MV4-11 cells transduced with a miR-10a or scrambled control mimic (H) were treated with a combination of Nutlin-3a and cytarabine at various doses and cell viability determined by MTS assay to quantify fractional inhibition of proliferation (Effect (Fa)).
  • the Combination Index (Cl) was calculated to determine antagonistic (Cl > 1), additive (Cl ⁇ 1), or synergistic (Cl ⁇ 1) effects between the two compounds. Nutlin-3a and cytarabine were strongly additive in OCI-AML3 cells but less so in MV4-11 cells.
  • Figure 4 provides results of an experiment designed to assess the effectiveness of a combination of Nutlin-3a, cytarabine and miR-10a inhibition on the survival time of animals of an aggressive AML model, namely mice provided with orthotopic xenografts established via tail vein injection of 5* 10 6 OCI- AML3 cells.
  • A mean survival 22.9 days vs 34.6 days; p ⁇ 0.001
  • B mean survival 27.4 days for miR-10a inhibitor vs 33.2 days for control inhibitor
  • p 0.02
  • the study aimed to determine whether a prediction of the response to such a treatment of AML could be made by measuring miR-10a expression and/or downstream genes/factors (eg TF ⁇ 20 ⁇ AP2C, TP53Ip53, RB1/ Rb, RBCCll RBCCl and in blood or bone marrow samples.
  • results described hereinafter indicate that, for example, a combination of an MDM2/MDMX inhibitor and an inhibitor of miR-10a, optionally with "standard of care” (SOC) treatment with the chemotherapeutic agent cytarabine (cytosine arabinoside or Ara-C), offers significant potential to provide a more effective treatment of AML and, particularly, those overexpressing miR-10a (ie a "miR-10a-high AML", including for example, ), especially in older subjects of 60 years of age or more.
  • SOC standard of care
  • this disclosure relates to a method of treating a haematological malignancy, preferably AML, or pre-malignant condition in a subject, said method comprising administering to said subject an effective amount of:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the method may be applied to the treatment of any haematological malignancy, such as a leukaemia, lymphoma, multiple myeloma and myelodysplastic syndromes (MDS), as well as pre- malignant conditions such as clonal haematopoiesis (CH).
  • haematological malignancy such as a leukaemia, lymphoma, multiple myeloma and myelodysplastic syndromes (MDS), as well as pre- malignant conditions such as clonal haematopoiesis (CH).
  • the method of the first aspect is applied to the treatment of AML. More preferably, the method is applied to the treatment of an AML overexpressing miR-10a (eg NPM C+ AML).
  • An AML overexpressing miR-10a ie a miR-1 Oa-high AML may be readily identified by those skilled in the art.
  • an AML subject characterised by miR-10a overexpression will show a level of miR-10a in a suitable body sample (eg a blood or bone marrow sample) that is higher than the typical or median level of miR-10a found in a comparable sample taken from a normal patient (eg a healthy patient with no apparent disease).
  • a suitable body sample eg a blood or bone marrow sample
  • Suitable assays for the determination of the level of expression of miR-10a include quantitative PCR-based assays known to those skilled in the art (eg Quantitative reverse transcriptase polymerase chain reaction (QRT-PCR), Real-time quantitative PCR (RT-PCT) and fluorescence in situ hybridisation (FISH); examples of specific QRT-PCT and RT-PCR assays for the determination ofmiR-10aare described in Zhi Y etal., 2015 [31] and Zhang T-j et al., 2018 [33]; the entire contents of which are both incorporated herein by reference).
  • QRT-PCR Quantitative reverse transcriptase polymerase chain reaction
  • RT-PCT Real-time quantitative PCR
  • FISH fluorescence in situ hybridisation
  • the subject may be characterised by miR-10a overexpression (ie such that the level of miR-10a present in a blood or bone marrow sample is above the typical or median level found in normal blood or bone marrow samples), and/or by modulation of downstream genes/factors (eg downregulation of any or all of and/or upregulation of
  • the method may be applied to the treatment of a subject characterised by high or overexpression of overexpression (ie such that the level of Mdm4 and/or mRNA present in a blood or bone marrow sample is above the typical or median level found in normal blood or bone marrow samples), especially when under genotoxic stress (ie conditions causing or likely to cause genotoxic (DNA) damage that may introduce mutations that may potentially generate other cancers; such conditions are well known to those skilled in the art and include, for example, treatment with chemotherapeutic drugs like Ara
  • the subject may further be characterised by the absence of the following biomarkers; loss of function mutations in the TP 53 and RB1 genes.
  • the subject may be selected on the basis of at least one of, but preferably all, of the following biomarkers: a high level of expression of miR-10a, absence of a loss of function mutation(s) in TP53, and the absence of a loss of function mutation(s) in RB1, as determined from one or more suitable sample(s). Loss of function mutations in TP53 and RB1 may be readily determined by DNA sequencing.
  • the method may be applied to the treatment of a subject with AML (especially, AML overexpressing miR-10a), at any or all of the induction (ie remission induction), consolidation and maintenance (ie post consolidation) phases of AML treatment [51],
  • the first agent comprising an inhibitor of MDM2 and/or MDMX may be selected from, for example, any of the MDM2/MDMX inhibitors known to those skilled in the art.
  • MDM2 inhibitor and MDMX inhibitor refer to agents which inhibit interaction between MDM2 and MDMX respectively, and p53.
  • MDM2/MDMX inhibitor encompasses agents that inhibit MDM2 selectively (ie agents that have little or no inhibitory action on MDMX; or in other words, have little or no opacity to inhibit the interaction between MDMX and p53), agents that inhibit MDMX selectively (ie agents that have little or no inhibitory action on MDM2; that is, have little or no capacity to inhibit the interaction between MDM2 and p53) as well as agents which inhibit both MDM2 and MDMX (so-called “dual inhibitors").
  • Suitable examples of MDM2/MDMX inhibitors include czs-imidazoline compounds, spiro- oxindole compounds, imidazothiazole, dihydroisoquinolinone, piperidine compounds and piperidinone [32],
  • the first agent is a nutlin compound (ie a cz.v-imidazoline compounds) such as Nutlin- 1, Nutlin-2 andNutlin-3 (ie ( ⁇ >4-[4,5-Bis(4-chlorophenyl>2-(2-isopropoxy- 4-methoxy-phenyl)-4,5-dihydro-imidazole-l-carbonyl]-piperazin-2-one) [62] [63], and especially the (-)- Nutlin 3 enantiomer known as Nutlin-3a, or a derivative or analogue thereof (eg ((4S,5R)-2-(4-(tert- butyl
  • MDM2/MDMX inhibitors include 3-(4- amino- 1-oxo 1 ,3-dihydro-2//-isoindol-2-y 1) piperidine-2, 6-dione (lenalidomide), (2'S,3R,4'S,5'R)-6- chloro-4'-(3-chloro-2-fluorophenyl)-2'-(2,2-dimethylpropyl)-l,2-dihydro-N-(/ra/w-4-hydroxycyclohexyl)- 2-oxo-spiro[3H-indole-3,3'-pyrrolidine]-5'-carboxamide (SAR405838), niraparib (MK4828), milademetan (DS-3032b), (S)-l-(4-chlorophenyl)-7-isopropoxy
  • the first agent may comprise two or more inhibitors of MDM2 and/or MDMX. Also, in some embodiments, the first agent may be a dual inhibitor such as the stapled a-helical peptides, ATSP-7041[38] and ALRN- 6924 [39]).
  • the second agent comprising an inhibitor of miR-10a may be selected from, for example, any of the miR-10a inhibitors known to those skilled in the art.
  • suitable inhibitors of miR-10a may take the form of a nucleic acid molecule (eg a polynucleotide or oligonucleotide molecule) which encodes interfering RNA (iRNA) targeted to miR-10a or constitutes an inhibitory RNA molecule itself (ie targeting miR-10a), or which provides a cis acting gene regulatory element to repress expression of miR-10a.
  • miR-10a inhibitors for use in the method of the first aspect are selected from anti-microRNA- 1 Oa agents comprising a single-stranded antisense oligonucleotide designed to specifically target mature miRNA 10a which are readily available (eg from Genepharma, Shanghai, China).
  • anti-microRNA- 1 Oa agents comprising a single-stranded antisense oligonucleotide designed to specifically target mature miRNA 10a which are readily available (eg from Genepharma, Shanghai, China).
  • General approaches to the design and synthesis of inhibitors to miRNAs are provided in International Patent Specification No WO 2005/079397, the entire content of which is incorporated herein by reference.
  • Other suitable examples of miR-10a inhibitors may include various small organic molecules as may be identified by, for example, screening of suitable compound libraries.
  • suitable miR- 10a inhibitors may include proteins which interact and inhibit miR-10a, as well as oligonucleotide- or peptide-aptamers which specifically bind to and inhibit miR-10a
  • the second agent may comprise two or more inhibitors of mi R- 10a.
  • the chemotherapeutic agent may be selected from, for example, any of the chemotherapeutic agents known to those skilled in the art, but preferably, from those chemotherapeutic agents that may be regarded as "standard” in the art or comprise what is considered a “standard of care” (SOC) chemotherapeutic agent.
  • SOC standard of care
  • Suitable standard chemotherapeutic agents may include agents belonging to the classes of alkylating agents (eg cisplatin and derivatives, nitrogen mustard compounds such as chlorambucil and busulfan, nitrosoureas including N-nitroso-N-methylurea (MNU) and carmustine (BCNU), and tetrazine compounds such as dacarbazine and temozolomide), anti-metabolites (eg antifolate compounds such as methotrexate (MTX)), fluoropyrimidines (eg 5-fluorouracil (5FU)), deoxynucleoside analogues (eg cytarabine and gemcitabine), and thiopurines (eg 6-thioguanine (6-TG) and mercaptopurine), anti-microtubule agents (eg Vinca alkaloids and taxanes), and topoisomerase inhibitors (eg the topoisomerase I inhibitors, irinotecan and topotecan, and agents
  • the chemotherapeutic agent is selected from cytarabine, anthracycline drugs, and combinations of such agents.
  • the chemotherapeutic agent is cytarabine (ie alone) or cytarabine and daunorubicin, which represent examples of standard of care treatments of AML.
  • the inclusion of an anthracycline drug such as daunorubicin may be preferably avoided as such drugs can show cardiotoxicity.
  • the first agent, second agent and chemotherapeutic agent may, for example, be administered to the subject concurrently (eg in combination) or all separately (ie consecutively) and in any order, or, for example, the first and second agents may be administered concurrently with the chemotherapeutic agent administered before or after, or the chemotherapeutic agent may be administered in combination with either of the first and second agents with the other agent (ie the "remaining" first or second agent as the case may be) administered before or after.
  • the first, second and chemotherapeutic agents are administered concurrently (ie in combination).
  • the respective agents when administered consecutively, may be administered one after another with practically no time interval (ie one is administered effectively immediately after the other) or, otherwise, after an interval of 1 to 5 minutes or more (eg 10 minutes, 30 minutes, 60 minutes, 4 hours or 12 hours between each of the agents).
  • the agents may each be formulated for administration in a pharmaceutical composition form including a pharmacologically acceptable carrier and/or excipient, which may be the same or different.
  • agents may be formulated for administration in a single pharmaceutical composition form (including a pharmacologically acceptable carrier and/or excipient) or as two pharmaceutical compositions (eg with a first pharmaceutical composition comprising the first and second agents, and a second pharmaceutical composition comprising the chemotherapeutic composition).
  • the second agent comprises a nucleic acid molecule (eg a single-stranded antisense oligonucleotide molecule designed to specifically target miR-10a), it may be preferred to formulate the agent so as to improve the efficacy of delivery or transfer to the subject's cells.
  • a nucleic acid molecule eg a single-stranded antisense oligonucleotide molecule designed to specifically target miR-10a
  • nucleic acid molecules may be complexed with cationic lipids to form cationic particles known as lipid nanoparticles (LNP), which may optionally be coated with, for example, an electrostatically adsorbed, poly(glutamic acid>based peptide coating to reduce toxicity and/or polyethylene glycol (PEG) to "shield” or neutralise the positive charges of the cationic particles (which can promote electronic association with negatively charged serum proteins, along with subsequent opsonisation and clearance of the particles).
  • LNP lipid nanoparticles
  • PEG polyethylene glycol
  • compositions suitable for use in administering the agents in accordance with the method of the first aspect may optionally comprise other substances such as absorption enhancers including surfactants (eg sodium lauryl sulphate, laureth-9, sodium dodecyl sulphate, sodium taurodihydrofusidate and poly oxyethylene ethers) and chelating agents (eg EDTA, citric acid and salicylates).
  • absorption enhancers including surfactants (eg sodium lauryl sulphate, laureth-9, sodium dodecyl sulphate, sodium taurodihydrofusidate and poly oxyethylene ethers) and chelating agents (eg EDTA, citric acid and salicylates).
  • the pharmaceutical composition ⁇ ) may be suitable for, for example, oral, buccal, sublingual, nasal, subcutaneous, intramuscular and intravenous administration.
  • the pharmaceutical composition ⁇ ) are particularly suitable for iv infusion.
  • the pharmaceutical composition(s) are particularly formulated for oral administration, and may therefore be provided in dosage forms such as compressed tablets, pills, capsules, caplets, gellules, and drops.
  • the pharmaceutical composition ⁇ ) will be administered to the subject in an amount which is effective for treating the disease (eg AML) and which, in the context of treating AML for example, may therefore result in one or more of the following: reduced leukaemic cell numbers, slowed spread of the disease, and amelioration of one or more AML disease symptom, increased survival, improved quality of life and cure rate for de novo secondary AML, treatment refractory and relapsed AML.
  • a composition comprising the first agent may be administered to provide, for example, an amount of tiie MDM2/MDMX inhibitor that is between about 0.1 and about 100 mg/kg body weight per day, or between about 5 and about 50 mg/kg body weight per day.
  • a composition comprising the second agent may be administered to provide, for example, an amount of the agent comprising an inhibitor of miR-10a that is between about 0.1 and about 100 mg/kg body weight per day, between about 5 and about 15 mg/kg body weight per day of the agent.
  • a composition comprising the chemotherapeutic agent will be administered to provide an amount of the chemotherapeutic agent that is in line with the typical (ie routine) amounts used for the particular chemotherapeutic agent in the clinic.
  • the method may enable the use of a relatively low dose of cytarabine (eg 20-50 mg per day compared to the standard dose of 100 to 200 mg per square meter per day) which may offer advantages such as reduced side-effects (eg ataxia, anaemia, GI disturbances, fever and dermatitis).
  • a relatively low dose of cytarabine eg 20-50 mg per day compared to the standard dose of 100 to 200 mg per square meter per day
  • advantages such as reduced side-effects (eg ataxia, anaemia, GI disturbances, fever and dermatitis).
  • the administered amount of the first and second agents and the chemotherapeutic agent, and the frequency of administration of any or all of these agents for any particular subject may vary and depend upon a variety of factors including the activity of the agents, the metabolic stability and length of action of the agents, the age, body weight, sex, mode and time of administration, and the rate of excretion of the agents.
  • composition(s) may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
  • the first agent and second agent are formulated in combination for administration in a single pharmaceutical composition.
  • This enables the consulting physician to readily treat the subject in accordance with the method of the first aspect by using the composition in combination with his/her preferred chemotherapeutic treatment (which may, of course, vary depending upon the characteristics of the subject (eg age) and the stage of the AML disease etc).
  • the combination composition may further comprise, for example, a pharmacologically acceptable carrier and/or excipient, optionally together with others substances such as absorption enhancers (including surfactants and chelating agents) and protease inhibitors (such as aprotinin (trypsin/chymotrypsin inhibitor), amastatin, bestatin, boroleucine, and puromycin (aminopeptidase inhibitors)) for improving bioavailability.
  • absorption enhancers including surfactants and chelating agents
  • protease inhibitors such as aprotinin (trypsin/chymotrypsin inhibitor), amastatin, bestatin, boroleucine, and puromycin (aminopeptidase inhibitors)
  • the subject will be a human.
  • the method of the first aspect may also be applicable to non-human subjects such as, for example, livestock (eg cattle, sheep and horses), exotic animals (eg tigers, lions, elephants and the like) and companion animals (such as dogs and cats).
  • livestock eg cattle, sheep and horses
  • exotic animals eg tigers, lions, elephants and the like
  • companion animals such as dogs and cats.
  • the method of the first aspect may further comprise a haematopoietic stem cell transplant (HSC Tx), especially for stronger and/or younger subjects (eg subjects younger than 60 years of age).
  • HSC Tx haematopoietic stem cell transplant
  • the first aspect of the present disclosure also relates to the use of:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • a chemotherapeutic agent for treating AML (preferably an AML overexpressing miR-10a) in a subject.
  • composition comprising:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity.
  • compositions of the second and third aspects may be suitable for, for example, oral, buccal, sublingual, nasal, subcutaneous, intramuscular and intravenous administration.
  • the composition may further comprise, for example, a pharmacologically acceptable carrier and/or excipient, optionally together with others substances such as absorption enhancers and protease inhibitors for improving bioavailability, especially oral bioavailability.
  • the composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
  • the present disclosure relates to a kit comprising first, second and third containers (eg vials), wherein the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX, the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity, and the third container contains a chemotherapeutic agent; optionally packaged with instructions for the use of the kit in the method of the first aspect
  • the present disclosure relates to a kit comprising first and second containers (eg vials), wherein the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX, and the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity; optionally packaged with instructions for the use of the kit in the method of the first aspect.
  • first and second containers eg vials
  • the first container contains a first agent comprising an inhibitor of MDM2 and/or MDMX
  • the second container contains a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the first agent second agent and chemotherapeutic agent provided in the containers of the kits may be as described above in relation to the method of the first aspect.
  • the kits may further comprise materials and agents for assaying for the determination of any or all of the biomarkers: miR-10a overexpression, high or overexpression (especially when under genotoxic stress), absence of a loss of function mutation(s) in TP53, and the absence of a loss of functionmutations) in RB1, as described above.
  • the present disclosure relates to a method of predicting effectiveness of MDM2 and/or MDMX inhibition in treating an individual with a malignant condition (preferably a haematological malignancy such as AML) or pre-malignant condition, comprising quantifying miR-10a expression and/or modulation of downstream genes/factors (eg downregulation of any or all of , and/or upregulation of in a suitable body sample (eg a blood or bone marrow sample).
  • a malignant condition preferably a haematological malignancy such as AML
  • pre-malignant condition comprising quantifying miR-10a expression and/or modulation of downstream genes/factors (eg downregulation of any or all of , and/or upregulation of in a suitable body sample (eg a blood or bone marrow sample).
  • the present disclosure relates to a method of treating a haematological malignancy or pre-malignant condition (preferably AML) in a subject, said method comprising: quantifying miR-10a expression and/or modulation of downstream genes/factors (eg downregulation of any or all of and/or upregulation of in a suitable body sample (eg a blood or bone marrow sample) to determine whether the subject is suitable for treatment with an inhibitor of MDM2 and/or MDMX; and treating the subject by administering an effective amount of:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • the subject is determined as being suitable for treatment with an inhibitor of MDM2 and/or MDMX by quantifying miR-10a expression at a level corresponding to a high level of expression of miR-10a, and/or by quantifying MDM4I Mdm4 expression at a level corresponding to a high level or overexpression of MDM4/Mdm4.
  • the present disclosure relates to the use of an effective amount of:
  • a first agent comprising an inhibitor of MDM2 and/or MDMX
  • a second agent comprising an inhibitor of miR-10a expression and/or activity
  • chemotherapeutic agent for treating a haematological malignancy, preferably AML, or pre-malignant condition in a subject.
  • Example 1 Targeting NPMl-mutant AML via reactivation of the p53/Rb tumour suppressor network
  • miR-10a may represent an oncomiR which contributes to the repression of the p53/Rb pathway by repressing key downstream pathway members including (but not limited to) TFAP2C, CDKN1 A, RB1CC1, T53 and MDM4.
  • key downstream pathway members including (but not limited to) TFAP2C, CDKN1 A, RB1CC1, T53 and MDM4.
  • the measurement of miR-10a expression in the context of AML may be used to predict how effective MDM2 inhibition may be in patients.
  • this therapeutic strategy could be enhanced by the simultaneous inhibition of miR- 10a to trigger the de-repression of p53/Rb-related targets.
  • miR-10a and negative control mi Script mimics and inhibitors were lentivirally-transfected into cultured cells.
  • the sequence of the miR- 10a inhibitor was: UACCCUGUAGAUCCGAAUUUGUG: SEQ ID NO: 1).
  • a p.L287*fs mutation was introduced into the GFP-NPM WT plasmid using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies Inc., Santa Clara, CA, United States of America) and suitable oligonucleotides.
  • CDKNla "Silencer Select" siRNA was obtained from ThermoFisher Scientific (assay s416; Waltham, MA, United States of America).
  • the CellTitre 96 Aqueous One Solution Cell Proliferation Assay (MTS) Assay (Promega Corporation, Madison, WI, United States of America) and Click-iT EdU Alexa Fluor 488 Flow Cytometry Cell Cycle Assay (ThermoFisher Scientific) were performed as per the manufacturer's instructions.
  • cDNA was prepared using the Superscript III First-Strand Synthesis SuperMix for qRT-PCR (mRNA; Life Technologies, Carlsbad, CA, United States of America) or the TaqMan MicroRNA Reverse Transcription Kit (miRNA; Applied Biosystems, Foster City, CA, United States of America) and measured using the SYBR Select Mastermix (mRNA; Applied Biosystems) or TaqMan Universal PCR Master Mix (miRNA; hsa-miR-10a assay ID 1093, mu6B assay ID 387; Applied Biosystems).
  • mRNA QPCR primer sequences are shown in Table 1 below. Table 1
  • Oligonucleotide sequences Primers used for QPCR (RB1CC1, TFAP2C, and ⁇ -Actm) and sile-direcled mutagenesis (NPM1-SDM).
  • Annexin V, LC3, p53, Rb, and ⁇ -Actin antibodies were obtained from Sigma-Aldrich (St Louis, MO, United States of America).
  • mice 5 ⁇ 106 OCI-AML3 cells were injected into the tail veins of 8- 12-week-old female mice. 12 days after cell delivery, mice were treated with cytarabine (50 mg/kg ip), Nutlin-3a (100 mg/kg oral gavage), a combination of both compounds at the dosages above, or a vehicle control 4 days/week for up to 3 weeks. Ethical endpoint was >20% weight loss or hind limb disability.
  • miR-1 Qa regulates several members of the p 53/Rb network miR-10a directly regulates the p53 regulator MDM4 [15], and also binds to the 3'UTR of two other key p53/Rb network genes: the p21 negative regulator TFAP2C and the Rb positive regulator RBI CC1 [12]. Since these effects may modulate response to MDM2 inhibition, an experiment was conducted to determine whether manipulating the intracellular availability of miR-10a would lead to the modulation of the expression of these genes and therefore, the activity of the p53/Rb network.
  • p53-/- KYO-1 cells transduced with a miR-10a mimic and similarly treated displayed no significant effects on target gene expression.
  • miR-10a-mediated modulation of TFAP2C its transcriptional target p21 was significantly downregulated (4-fold, p ⁇ 0.05 for both).
  • Sensitivity to MDM2 inhibitor Nutlin-3a is regulated bv downstream miR-10a gene targets Activation of the p53 network in response to cell damage can promote cytoprotective cell cycle arrest and autophagy or cytotoxic apoptosis depending on the cellular context, with the former responses representing a major determinant of resistance to a wide variety of therapeutic compounds in cancer [20], miR-10a downstream targets p53 (TP53), Rb (RB1) and p21 (CDKN1A) have central roles in this response network.
  • TP53 p53
  • Rb RB1
  • CDKN1A p21
  • miR-lQa and/or its downstream targets are biiomarkers of MDM2 inhibitor and cvtarabine responsiveness in AML patient samples
  • miR-10a was measured in diagnostic samples immediately prior to treatment and correlated to outcome.
  • SAL Study Alliance Leukemia
  • HSCT allogeneic hematopoietic stem cell transplantation
  • miR-10a mediates response to Nutlin-3a and cvtarabine in AML
  • miR-10a-high AML cell lines OCI-AML3 and IMS-M2 were transduced with a miR-10a inhibitor (Qiagen NV), which resulted in a significant sensitisation to both compounds ( Figure 3A and 3B).
  • a miR-10a inhibitor Qiagen NV
  • resistance to both compounds could be induced by overexpressing miR-10a in miR-10a-low AML cell lines MV4-11 and HL60 ( Figure 3C and3D).
  • Activation of the p53/Rb-mediated stress response is potentiated by increased expression of miR-10a target RBCC1 via its direct binding to and stabilisation of p53, and subsequent upregulation of Rb, pl6, and p21 [18],
  • Excessive accumulation of Rb and p53 in the cytoplasm represses pro-survival autophagy and instead promotes intrinsic apoptosis via their direct interaction with Bcl-2 family proteins [23]
  • significantly fewer miR-10a- inhibited OCI-AML3 cells had detectable autophagic vacuoles as determined by monodansylcadaverine (MDC [24]) fluorescence following treatment with Nutlin-3a or cytarabine.
  • MDC [24] monodansylcadaverine
  • Cytotoxic stress leading to the activation of cytoprotective autophagy is universally associated with cell cycle arrest mediated by downstream miR-10a target p21 [25], Extended arrest gives the cells sufficient time to repair DNA and recycle organelles before recommencing proliferation, and an inability to do so results in the accumulation of DNA damage and mitotic catastrophe leading to apoptosis [26] [27], OCI-AML3 cells transduced with a scrambled control inhibitor and treated with Nutlin-3a or cytarabine arrested in G0/G1 -phase and S-phase, respectively, as has been previously observed [28] [29].
  • a combination of Nutlin-3a. cvtarabine and miR-10a inhibition is an effective therapeutic strategy for miR-10a-high AML in vivo
  • miR-10a-high AML orthotopic xenografts were established via tail vein injection of 5> ⁇ 10 6 cells transduced with a miR-10a or scrambled control inhibitor, resulting in rapid engraftment of aggressive acute leukaemia with a mean survival of just 22 days.
  • mice 12 days after cell delivery (ie to allow for engraftment of the cells and an aggressive acute leukaemia to develop), mice were treated with cytarabine, Nutiin-3a, a combination of the compounds, or a vehicle control 4 days/week for up to 3 weeks.
  • Inhibition of miR-10a markedly improved the survival of animals treated with Nutlin-3a compared to the control inhibitor group (mean survival 22.9 days vs 34.6 days; p ⁇ 0.001; Figure 4A).
  • Combination treatment modalities particularly those that target multiple nodes of the same network, have the potential for increased efficacy, decreased toxicity, and reduced likelihood of drug resistance.
  • MDM4 and TP53 were consistently among the top genes that made up the predictive signatures). Independent validation is required to confirm the power of these panels to ensure model overfitting has not occurred however - an attempt to validate the 4-gene signature in a subsequent study for example [43] demonstrated a PPV no better than using TP53 mutation status itself [25],
  • miR-10a may also be attractive as a biomarker of cytarabine-based therapy with which MDM2 inhibition has shown synergy [44], and can be effectively targeted therapeutically using miRNA inhibitors to sensitise to the combination therapy that has been demonstrated here.
  • MDM2 inhibitors such as Nutlin-3a triggers the massive intracellular accumulation of p53 resulting in the activation of one (or sometimes both) of the tumour suppressors’ contrasting canonical signalling networks - either cytoprotective, promoting cell cycle arrest, autophagy and DNA repair, or cytotoxic, promoting cell death via the activation of apoptosis [37] [45], Cell fate depends heavily on the status of key nodes within these signalling networks [46], of which four - p21, Rbl, MDM4 and p53 itself - are shown here to be both downstream targets of miR-10a as well as the most important determinants of cellular sensitivity to both Nutlin-3a and cytarabine.
  • miR-10a regulates cell cycle inhibitor p21 by directly binding to and downregulating its repressor TFAP2C [13], with miR-10a inhibition ameliorating the ability of p53 to upregulate p21 in response to genotoxic stress, coinciding with an inability to initiate cell cycle arrest and the subsequent activation of apoptosis.
  • p21 antisense therapy has proven to be a viable adjuvant to DNA damaging therapeutics via its promotion of apoptosis in preclinical in vivo models [47], and helps explain the synergy between Nutlin-3a and miR-10a inhibition observed here.
  • the upregulation of TFAP2C by miR-10a inhibition similarly synergises with Nutlin-3a to tip the cellular balance away from autophagy and towards apoptosis.
  • miR-10a also directly targets RB1CC1 [13], which plays an essential role in the induction of autophagy in mammalian cells [48].
  • Autophagy is an evolutionarily-conserved mechanism activated in response to cell stress that acts as a buffer against damaging stimuli, in which damaged cytosolic components and organelles are degraded and recycled through the lysosomal machinery [49], The activation of autophagy therefore normally results in resistance to weak-to-moderate cytotoxic stressors, including sublethal concentrations of chemotherapeutic agents [50],
  • autophagy is universally associated with cell cycle arrest, requiring the induction of p21- mediated cellular senescence for successful execution [34],
  • loss of p21 coincided with the inhibition of autophagy activation, as has been observed previously [51]
  • RB1CC1 is also central to p53-mediated damage sensing and like p53 plays a dual role in the response to cell stress.
  • RB1CC1 switches from its canonical role as an inducer of cytoprotective autophagy to one of classical tumour suppressor and apoptosis inducer.
  • This fundamental switch evolutionarily conserved from nematodes to mammals, is primarily determined by the abundance of p53 in the cytoplasm.
  • miR-10a target MDM4 shown here to be upregulated in primary AML cells sensitive to the MDM2 inhibitor DS-3032b as well as in samples from responders in a clinical trial, exhibits pleotropic effects depending on cellular context.
  • MDM4's canonical role in non-stressed cells is to cooperate with the central p53 inhibitor MDM2 to enhance its E3 ubiquitin ligase activity which marks p53 for degradation.
  • it can stabilise the secondary structure of the TP53 mRNA via interaction with its C-terminal RING domain, which conversely promotes the induction of p53 synthesis.
  • MDM2 can compete with MDM2 for p53 binding which stimulates p53 stabilisation and relocalisation to the cytoplasm [17] where p53 can further contribute to apoptotic activation.
  • AML cells that express high endogenous MDM4 levels under genotoxic stress can be highly sensitive to MDM2 inhibition [17]
  • MDM2 similarly promotes ubiquitin-dependent degradation of the Rb protein [35] in a process which is inhibited by competitive binding by MDM4, resulting in the activation of the Rb network when MDM4 is upregulated [36]
  • this interaction is likely to be dependent on cellular context, and promoted in response to genotoxic stress.

Abstract

La présente invention concerne un procédé de traitement d'une malignité hématologique ou d'un état pré-malin chez un sujet. Dans une application particulière, le procédé comprend l'administration à un sujet atteint d'une leucémie myéloïde aiguë (AML) et de préférence une AML surexprimant miR-10a (eg NPMc+ AML), une quantité efficace d'un premier agent comprenant un inhibiteur de MDM2/MDMX (par exemple, la nutline -3a), un second agent comprenant un inhibiteur de l'expression et/ou de l'activité de MiR-10 (par exemple un oligonucléotide antisens ciblant MiR-10a), et éventuellement, un agent chimiothérapeutique tel qu'un agent chimiothérapeutique "standard of care" (SOC) pour traiter l'AML (par exemple cytarabine).
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